Fluoroquinolones are the most frequently prescribed antimicrobial agents in the United States. Fluoroquinolone- and multidrug-resistant bacteria are emerging infectious disease agents of worldwide concern and E. coli is an important human pathogen and excellent model organism. The long-term goals of the Zechiedrich laboratory are to determine how bacteria respond to and resist antimicrobial agents and to use this knowledge both to prolong the usefulness of current drugs and to aid in the design of new therapeutic protocols to help prevent and combat fluoroquinolone- and multidrug-resistant bacterial infections. The goals of this competing renewal proposal are to determine the (i) regulation, (ii) cellular roles, and (iii) molecular mechanisms of the multidrug efflux pumps, and the (iv) genetic alterations and (v) selection mechanisms that cause increased drug MICs in patient isolates. With thousands of characterized and uncharacterized fluoroquinolone- and multidrug-resistant E. coli patient isolates and the accompanying patient data, >9 million patient visits/year in the Texas Medical Center, and the strength of the combined expertise of the investigators, the goals will be achieved with the following Specific Aims: (1) Understand the interplay between and molecular mechanisms of the efflux pumps important for fluoroquinolone-resistance in E. coli; and (2) Identify molecular mechanisms, mutant selection mechanisms, and genetic pathways to fluoroquinolone- and multidrug-resistance in E. coli clinical isolates. These Specific Aims will be achieved by three highly inter-related approaches: (i) A molecular approach will determine the transcriptional relationships among the fluoroquinolone efflux pumps and their regulators using quantitative reverse- transcription PCR first in defined mutant strains and then in fluoroquinolone-resistant E. coli clinical isolates; (ii) A computational approach will uncover evolutionarily related regions of the efflux pumps indicative of functionality; and (iii) A genomic approach using cost effective sequencing technologies will identify relevant heritable variations that correlate with the drug-resistance of the clinical isolates. These approaches are tied together using bioinformatics and data management infrastructures. PUBLIC HEALTH RELEVANCE: With few new antimicrobial agents being developed, we must work to preserve those used currently. Excellent bioavailability, low toxicity and, in general, low resistance frequency, make the fluoroquinolones an important drug class to maintain in the physician's arsenal. Results from the proposed translational research will aid in the prescription practices of physicians and in the design of new therapeutic protocols to combat drug-resistant bacterial infections.